1
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2
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3
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- Point mutation – substitution T®G (see genetic code, page 111)
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4
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- Commentary - protooncogene c-ras
- G protein ® GTP-binding
protein with signal transmitting function
- Activated after binding growth factor to its receptor
- Activation of receptor ® exchange of associated GDP to GTP=
li>
- GTP triggers a short time-limited Ras signaling ability
- Signaling pathway continues via MAP-kinases
- In the nucleus ®
activation of transcription factors (e.g. product of c-myc)=
li>
- Result:
- c-ras is inactivated after short time by conversion of GTP to GDP
- Sev=
eral
mutations in c-ras can remove the time limit of the cell-stimulated
signal (hydrolysis of =
GTP to
GDP) and uncontrolled cell division leads to tumor development
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5
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- NORMAL GENES – CELL CYCLE REGULATION ®=
font> PREVENTION OF NEOPLASIA
- MUTATION ®
loss of gene function
- MUTATION RECESIVE AT T=
HE
CELLULAR LEVEL (loss of function of both alleles)
- AUTOSOMAL DOMINANT INHERITANCE
- HEREDITARY PREDISPOSITION ® GERMINAL MUTATION
- (AT THE LEVEL OF WHOLE ORGANISM)
- Sporadic incidence of tumor: two somatic mutations ® risk =3D frequency neoplasia in population=
- Familial incidence: 1. germline mutation, 2. somatic mutation
- predisposition, early childhood, multifocal or bilateral incidence, =
several
memmbers of family affected
- tumor initiation - requires two steps (Knudson – two-hit
hypothesis)
- loss of heterozygosity in tumor cells (LOH) – e.g. linkage
analysis Rb1 gene with gene coding enzyme esterase D (marker)
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6
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- Deletion – could be found in different locations of a gene
- Mitotic nondisjunction
- Mitotic recombination
- Uniparental disomy (both chromosomes originate from the same parent)
- Point mutation
- Protein inactivation (interaction with viral antigene)
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7
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8
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- RECESIVE MUTATION
- FIRST MUTATION – GERMLINE
- SECOND SOMATIC
- Two-hit hypothesis
- (Knudson)
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9
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10
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- Rb1 gene – ubiquitously expressed
- Cell cycle regulation
- gene product – nuclear phosphoprotein (pRb - 100kD)
- phosphorylation / dephosphorylation (serine and threonine residues)<=
/li>
- dephosphorylation of protein Rb – active form ® inactivation of transcriptional factor E2F=
- ® cell=
cycle
block in G1
- phosphorylated pRb (inactive) ® E2F released from complex pRb/E2F=
li>
- transition fom G1 to S phase of interphase
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11
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12
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13
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14
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- Products BRCA genes form complex with products of several genes
- Cell cycle regulation
- Repair – doublestrand breaks of DNA
- Inherited mutation of BRCA1 – familial incidence breast cancer
and/or cancer of ovary
- Germline mutation of BRCA2 gene – association with cancer of
breast (both female and male), prostate and pancreas
- proteine BRCA1 and BRCA2 ® cooperation with protein RAD51
- Mutation of BRCA1 / BRCA2 gene – truncated protein (loss of
function)
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15
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16
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17
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18
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19
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- Association with  =
;HNPCC
(Hereditary Nonpolyposis Colon Cancer)
- hMSH2, hMLH1, hPMS1, hPMS2, hMSH6, hMSH3
- genome stability
- mismatch repair (replication errors)
- microsatellite loci (e.g. CAn) instability
- increased mutation frequency 100 –1000x
- recessive character of
mutation in MMR genes
- autosomal dominant heritability HNPCC
- Lynch syndrome I – colon and rectal cancer
- Lynch syndrome II - cancer of colon and rectum, 30% - tumor endometrium, stomach, pancr=
eas,
urinary tract
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20
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21
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- Nonrandom (primary)
- Philadelphia chromosome (Ph1)
- Translocation – Burkitt lymphoma
- Amplification, double-minutes
- Random (secondary)
- random chromosomal aberrations (deletions, translocations, dicentric
chromosome, ring chromosome, isochromosome ….)
- heteroploidy, for example pseudodiploidy
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22
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23
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24
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25
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26
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- FISH – diagnostic tool of clasical cytogenetics combined with
molecular genetic approach
- Based on hybridization of single stranded DNA probe with single stra=
nded
sequence of studied DNA in situ (based on the complementarity rule)<=
/li>
- Examination of chromosomes in mitotis or interphase
- DNA probe is labeled with fluorochrome (eg. Texas Red, FITC, etc.)=
li>
- Types of DNA probes – centromerical, locus-specific, painting =
of
chromosomes
Centromeric probes: a-satellite sequences of repetitive DNA pres=
ent in
centromeric regions ® detection of abnormal chromosome number
- Locus-specific probes: hybridization with specific loci on chromoso=
mes ® detection =
of
structural aberrations (microdeletion, translocations…)=
- Painting of whole chromosomes: mix of chromosome-specific DNA fragm=
ents
enables to distinguish different chromosomes, but cannot be used for
interphase chromosomes
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27
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28
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29
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30
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31
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32
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